Flow cytometry, buoyant density-gradient cell fractionation, dissociated cell culture, cell migration, immunocytochemistry and in situ hybridization methods are applied to embryonic/early postnatal rat CNS tissues to study the development, differentiation and cellular distribution of transmitter, transmitter-related enzymes and their corresponding receptors. During the past several years, we have focused primarily on GABA, which is transiently expressed in a widespread manner during CNS development before it becomes relatively restricted to fast- transmitting synapses in the adult where it often functions in an inhibitory manner. In FY95 we investigated the following: 1) transcripts encoding three GABA-synthesizing GAD enzymes and those encoding most GABA/A receptor subunit proteins were detected by in situ techniques in progressively more regions of the developing CNS; 2) specific subunits are expressed at all levels of the embryonic neuraxis beginning during the period of intense neuroblast proliferation; 3) distinct patterns of GAD and GABA transcript coexpressions are apparent: one almost exclusively in cells of the neuroepithelial proliferative zone, one in many, if not most differentiating cells during embryogenesis and one differentiating during the postnatal period; 4) some transcripts are only transiently detected for variable periods while others persist, becoming restricted to subpopulations; 5) transcripts encoding GAD and GABA receptors are more abundant and widely distributed during embryogenesis than in the adult, implying "morphogenic" roles; 6) GAD and GABA receptor subunit family proteins were detected in cells and processes, indicating that the transcripts are functional; 7) continuous-gradient centrifugation of embryonic CNS cells reveals that subpopulations accumulate in visible bands of specific buoyant density beginning during the logarithmic growth period; 8) FACS analyses of fractionated cells show that highly proliferative cells populate the dense region while primarily post-mitotic elements in G0 or G1 compose the visible bands; 9) FACS studies show that differentiation marker+, GAD+, GABA+, GABA/A receptor subunit+ cells exhibit characteristic patterns of expression according to their buoyant density; 10) GABA+ immunoreactivity can be eliminated by ionophore treatment, indicating dynamic lability in the immunodetectable signals; 11) FACS recordings of membrane potential and Cac2+ levels reveal characteristic patterns of functional GABA, glutamate and ACh receptor-coupled responses exhibited by different subpopulations cortical cells show that gradient-directed migration occurs at femtomolar GABA, while gradient-independent motility requires micromolar GABA; 13) structure-activity studies show that A, B and C are implicated; 14) GABA loaded with BAPTA-AM, indicating Cac2+-dependent signal transduction; 15) GABA+ spinal and hippocampal neurons differentiate in culture independently of the initial presence of cortical astrocytes.